The long-term objective of this project is to develop an understanding of the acute modulation of dopamine (DA) transporter (DAT) function by amphetamine (AMPH) in vitro and in vivo. DA signaling at CNS synapses regulates a variety of cognitive, emotional and behavioral functions. Abnormalities in the DA system have been implicated in a number of psychiatric and neurological disorders, including drug addiction, schizophrenia and Parkinson's disease. AMPH induces reverse transport of DA, thereby increasing extracellular DA levels and leading to its psychostimulant effects. To date, efforts to investigate AMPH regulation of DAT function have largely relied on traditional biochemical and/or biophysical approaches, often in heterologous expression systems. Although these efforts have revealed many novel aspects of AMPH actions, it has been difficult to establish a single system in which to manipulate the AMPH regulation of DAT function within a neuronal environment This project will combine molecular, biochemical and biophysical approaches to characterize AMPH regulation of DAT function both in neuronal preparations and in vivo. In this proposal, we will create a novel experimental platform to study DAT protein-protein interactions using the transgenic and experimental tools offered by Caenorhabditis elegans. The key issues to resolve include how DAT protein associations with the SNARE protein UNC-64 (the C. elegans homolog of syntaxin 1A) are essential for AMPH-induced DA efflux and AMPH-induced behaviors. Our experimental plan links the AMPH-induced functional regulation of DAT to stimulation of animal behaviors. The proposed studies address the following Specific Aims: 1) To create transgenic worm lines with impaired DAT/UNC-64 interaction. 2) To determine whether DAT/UNC-64 interactions regulate DAT-1 function and DAT-1- mediated AMPH effects in vivo and in vitro. The goal is to generate an experimentally-based model advancing our understanding in vitro and in vivo of the AMPH actions and to discover novel pathways and molecules that may contribute to disrupted DA signaling in disease states such as addiction and Parkinson's disease. PUBLIC HEALTH RELEVANCE: The dopamine transporter (DAT), the protein which carries out the DA reuptake process at the plasma membrane, is the major molecular target of several psychoactive drugs, including amphetamine (AMPH) and cocaine. Abnormalities in the dopaminergic system have been implicated in a number of psychiatric and neurological disorders, including drug addiction, schizophrenia and Parkinson's disease. The major goal of this proposal is to create an in vivo expression system to study DAT-interacting proteins and their effects on AMPH-induced DA efflux with the intent of elucidating how these proteins support AMPH-induced behaviors in living animals.